Behavior of 2, 6-Bis (decyloxy) naphthalene Inside Lipid Bilayer

J. Phys. Chem. B 2010, 114, 15483–15494

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Behavior of 2,6-Bis(decyloxy)naphthalene Inside Lipid Bilayer Mariusz Kepczynski,*,† Marta Kumorek,† Michał Stepniewski,† Tomasz Ro´g,‡ Bartłomiej Kozik,† Dorota Jamro´z,† Jan Bednar,§,⊥ and Maria Nowakowska*,† Faculty of Chemistry, Jagiellonian UniVersity, Ingardena 3, 30-060 Krako´w, Poland; Department of Physics, Tampere UniVersity of Technology, PO Box 692, FI-33101 Tampere, Finland; Laboratoire de Spectrome´trie Physique, UMR 5588, CNRS/UJF, BP87, 140 AV. de la Physique, 38402 St. Martin d’Heres Cedex, France; and First Faculty of Medicine, Institute of Cellular Biology and Pathology, Charles UniVersity in Prague, and Department of Cell Biology, Institute of Physiology, Academy of Sciences of the Czech Republic, V.V.i., AlbertoV 4, 128 01 Prague 2, Czech Republic ReceiVed: April 26, 2010; ReVised Manuscript ReceiVed: October 12, 2010

Interactions between small organic molecules and lipid or cell membranes are important because of their role in the distribution of biologically active substances inside the membrane and their permeation through the cell membranes. In the current paper, we have explored the effect of the attachment of long hydrocarbon tails on the behavior of small organic molecule inside the lipid membrane. Naphthalene with two decyloxy groups attached at the opposite sites of the ring (2,6-bis(decyloxy)naphthalene, 3) was synthesized and incorporated into phosphatidylcholine (PC) vesicles. Fluorescence methods as well as molecular dynamic (MD) simulations were used to estimate the position, orientation, and migration of compound 3 in PC bilayer. It was found that the naphthalene ring of compound 3 resides in the upper acyl chain region of the bilayer and the hydrocarbon tails are directed to the center of the bilayer. As was shown with cryotransmission electron microscopy (cryoTEM), such lipidlike conformation enables compound 3 to be incorporated into liposomes at a very high content without their disintegration. Moreover, compound 3 can migrate from one leaflet to other. The mechanism of this process is, however, different from that characteristic of the flip-flop event of lipid molecules in the membrane. Finally, the possible application of compound 3 as a rotational molecular probe for monitoring fluidity of liposomal membrane in the acyl side chain region was checked by studies of the effect of cholesterol on the fluorescence anisotropy of 3. 1. Introduction Interactions between small organic molecules and lipid and/ or cell membranes are important due to the crucial role of such interactions in the processes of drugs and nutrients uptake, and passive transport of the small molecules through the cell membranes. These interactions have also been explored due to their relevance in applications, such as molecular probes for monitoring of the organization and dynamics of the membranes, drug loading and release from liposomes,1,2 and, recently, drug overdose treatment with liposomes.3,4 The lipid bilayer is very heterogeneous in the normal direction, with large gradients in density and polarity on a nanometer length scale.5 Therefore, the bilayer can be divided into four arbitrary regions:5 (i) region I of the lowest density contains almost exclusively the lipid tails (spread 0-10 Å from the center of the bilayer), (ii) region II contains a diverse mixture of functional groups, mostly the carbonyl groups and a portion of the head groups, and is effectively the interface between the hydrocarbon chain region and the polar region (10-18 Å from the center of the bilayer), (iii) region III contains the bulk of the headgroups and a substantial amount of water (18-25 Å from the center of the * Corresponding author. Tel.: +48 12 6632250. Fax: +48 12 6340515. E-mail: [email protected] (M.N.); [email protected] (M.K.). † Faculty of Chemistry, Jagiellonian University. ‡ Department of Physics, Tampere University of Technology. § Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague. ⊥ Laboratoire de Spectrometrie Physique, CNRS.

bilayer), and (iv) region IV consists mostly of bulk water, with small amounts of the headgroups (>25 Å from the center of the bilayer). These four arbitrary regions for an 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) membrane are illustrated in Figure s1 (see Supporting Information). Methods based on the fluorescence measurements have been used as the main tools to study the lipid bilayer structure and association of small molecules with the membranes. Using these methods, one can experimentally determine several important parameters: (i) equilibrium partitioning (binding) of the molecule to the lipid bilayer can be estimated using fluorescence titration technique,6,7 (ii) position of the molecule in the membrane can be determined using depth-sensitive fluorescent quenching analyses,8-10 (iii) the effect of the molecules on the membrane properties, such as liposome permeability, polarity, organization, and dynamics of lipids, can be determined using fluorescing molecular probes,3,11,12 and (iv) the lateral diffusion coefficients of lipid-soluble molecules can be estimated by fluorescence spectroscopy.13 Computer simulations provide a complementary view to experiments, revealing a level of detail that is often impossible or very difficult to achieve experimentally.5 There are two excellent reviews on the application of molecular dynamics (MD) simulations in studies of the interaction of small molecules with lipid bilayer. The first review is focused on the partitioning of small molecules as it pertains to liposomal drug transport,14 whereas the second one reports on the permeation of small molecules through membranes and their influence on the properties of lipid bilayers.5 Interactions of several aliphatic and

10.1021/jp103753f  2010 American Chemical Society Published on Web 11/08/2010

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aromatic molecules with lipid membranes were previously studied using MD simulations. MacCallum and Tieleman have studied partitioning of hexane into a dioleoylphosphatidylcholine model membrane.15 It was shown that hexane partitions preferentially to the center of the bilayer, in the disordered hydrocarbon tails of region I. Benzene and pyrene are the most intensively studied examples of the hydrocarbon aromatic compounds. Norman and Nymeyer have studied the localization and orientation of benzene inside a POPC membrane.16 They found three regions in the bilayer in which benzene is preferably localized. The most stable location is that of the hydrocarbon core (region I). There are two weaker binding sites: one near the glycerol moiety (region II) and the other one near the choline moiety (region III). Two computational studies have attempted to address the behavior of free pyrene in the lipid bilayer. Hoff et al.17 have shown that pyrene accumulates inside the lipid membrane near the interfacial region (region II) and the long axis of the pyrene molecules is preferentially directed parallelly to the lipid tails. Curdova et al.18 have studied free pyrene inside gel- and fluid-like phospholipid membranes. They have also found that pyrene molecules prefer to be located within the hydrophobic acyl chain region, close to the glycerol group of lipid molecules (region II). Their orientation though depends on the phase of the membrane. In the fluid phase, pyrene favors the orientation parallel to the membrane normal, while, in the gel phase, the orientation is affected by the tilt of the lipid acyl chains. In the current paper, we present complementary results of fluorescence and MD computer simulations studies on the effect of attachment of long hydrocarbons tails on the behavior of small organic molecule inside the lipid membrane. For that purpose, the new compound, 2,6-bis(decyloxy)naphthalene (3), was synthesized. Fluorescence studies and MD computer simulations were used to study the position and orientation of molecule 3 inside the lipid bilayer and possibility of a translocation (flipflop) of compound 3 from one monolayer to the other. CryoTEM method was used to observe the effect of 3 on the liposome vesicle formation. Finally, usefulness of 3 as a molecular probe to study properties of lipid bilayer modified by the addition of cholesterol was determined. 2. Materials and Methods 2.1. Materials. L-R-Phosphatidylcholine type XIII-E from egg yolk (EYPC, 99%, solution of 100 mg/mL in ethanol) was obtained from Sigma Chemical Co. (St. Louis, MO). It was a mixture of lipids with the following fatty acid makeup: 33% C16:0 (palmitic), 13% C18:0 (stearic), 31% C18:1 (oleic), and 15% C18:2 (linoleic) (other fatty acids being minor contributors), which gives an average molecular weight of approximately 768 g/mol. Poly(methyl methacrylate) (PMMA), cholesterol (Chol), naphthalene-2,6-diol (1), 1-bromodecane (2), and anhydrous DMF (99.8%) were purchased from Sigma-Aldrich. Stable free radicals 16-doxylstearic acid (16-SASL), 5-doxylstearic acid (5-SASL), and 3β-doxyl-5R-cholestane (CSL) as well as brominated carboxylic acids 2-bromohexadecanoic acid (2-Br), 16-bromohexadecanoic acid (16-Br), and 11-bromoundecanoic acid (11-Br) were purchased from Aldrich. 12Doxylstearic acid (12-SASL) was received from Molecular Probes (Eugene, OR). K2CO3 (pure p.a.) was received from Chempur and was dried in a vacuum oven. All solvents were obtained from POCH (Gliwice, Poland) and were of spectroscopy grade. Milipore-quality water was used during the experiments. 2.2. Synthesis of 2,6-Bis(decyloxy)naphthalene (3). Synthesis of 3 is schematically presented in Figure 1. Compound 1

Kepczynski et al.

Figure 1. Synthesis of 2,6-bis(decyloxy)naphthalene (3) and the atom numbering as used throughout the text.

(0.80 g, 4.99 mmol), dry DMF (50 mL), and K2CO3 (1.72 g, 12.48 mmol) were placed in a two-neck round-bottom flask, equipped with the dropping funnel and drying tube with anhydrous CaCl2. The mixture was heated up to 85 °C and the solution of 2 (2.21 g, 12.48 mmol) in 35 mL of dry DMF was added dropwise during 20 min. After stirring at 85 °C for 12 h a second portion of K2CO3 (1.72 g, 12.48 mmol) was added and the reaction was continued for further 4 h. Then the reaction mixture was stirred at room temperature for ca. 20 h. The reaction progress was monitored by the TLC (toluene/SiO2), which after 12 h of the reaction indicated only traces of 1. The samples for the TLC were prepared by dissolving 2-3 drops of the reaction mixture in ca. 2 mL of water followed by extraction with ca. 2 mL of toluene. After completion of the reaction, DMF was evaporated under reduced pressure, and water (50 mL) was added. The mixture was neutralized with 10% HCl and extracted with toluene (3 × 40 mL). Combined organic layers were washed with water (50 mL) and brine (50 mL) and dried over anhydrous MgSO4. Toluene was evaporated under vacuum. Crude product was purified by column chromatography (CCl4/SiO2) and recrystallized from MeOH to give 0.75 g (33.9%) of 3 as white plates, mp 82-83.5 °C. IR (KBr) νjmax (cm-1) 2957, 2935, 2920, 2851, 1606, 1509, 1467, 1395, 1380, 1244, 1169, 1159, 1115, 1020, 848, 813. 1H NMR (300 MHz, CDCl3, δ) 0.88 (t, J ) 6.7 Hz, -CH2CH3, 6H), 1.27-1.54 (m, -CH2-, 28H), 1.78-1.87 (m, -OCH2CH2-, 4H), 4.03 (t, J ) 6.6 Hz, -OCH2-, 4H), 7.07 (d, J ) 2.5 Hz, C1-H, C5-H, 2H), 7.11 (dd, J ) 8.7 Hz, J ) 2.5 Hz, C3-H, C7-H, 2H), 7.60 (d, J ) 8.7 Hz, C4-H, C8-H, 2H). 13C NMR (75 MHz, CDCl3, δ) 14.10 (2C, CH3-), 22.68 (2C, CH3CH2-), 26.13 (2C), 29.32 (4C), 29.43 (2C), 29.57 (2C), 29.60 (2C), 31.90 (2C), 68.08 (2C, -OCH2-), 106.98 (2C, C-1, C-5), 119.18 (2C, C-3, C-7), 127.99 (2C, C-4, C-8), 129.71 (2C, C-9, C-10), 155.54 (2C, C-2, C-6). Anal. Calcd for C30H48O2: C, 81.76; H, 10.98. Found: C, 81.75; H, 11.13. 2.3. Apparatus. 1H and 13C NMR spectra were taken on a Bruker Avance II 300 instrument at 300 and 75 MHz, respectively, using tetramethylsilane as internal standard. IR spectrum was recorded on Bruker IFS 48FT spectrophotometer. Elemental analysis was performed on a EuroEA 3000 Elemental Analyzer. UV-vis absorption spectra of the sample were measured in room temperature using a Varian Cary 50 spectrophotometer with combined halogen and deuterium light source. The spectra were recorded in a wavelength range of 200-850 nm. Steady-state fluorescence spectra of the samples were recorded on a SLM-AMINCO 8100 Instruments spectrofluorimeter equipped with a refrigerating and heating circulator Julabo F25. Emission spectra were corrected for the wavelength dependence of the detector response by using an internal correction function provided by the manufacturer.

2,6-Bis(decyloxy)naphthalene Inside Lipid Bilayer

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2.4. Preparation of Small Unilamellar Liposomes (SUV). Phospholipid vesicles containing compound 3 and Chol were prepared by sonication. Chol and 3 were first dissolved in ethanol and CHCl3 to form stock solutions. Appropriate volumes of the stock solutions were combined with 100 µL of the lipid solution in a volumetric flask, and the solvents were evaporated under flow of nitrogen. The dry material was dissolved in diethyl ether which was then reevaporated under nitrogen to complete dryness. Ten millimoles of phosphate buffer was added until the desired lipid concentration was attained (usually 2.5 mg/ mL), and the sample was vortexed for 2 min. The resulting multilamellar vesicle dispersion was subjected to five freeze-thaw cycles from liquid nitrogen temperature to the temperature of 60 °C, followed by sonication at ice temperature for 10 min using a titanium tip SONICS VC 130. To remove titanium particles coming from the sonicator horn the received SUV suspension was centrifuged at 8000 rpm for 10 min. 2.5. Fluorescence Quantum Yield Measurements. The fluorescence quantum yield (Q) of 3 in various solvents was determined by using the expression19

Q ) QR

I 1 - 10-AR n2 IR 1 - 10-A n 2

(1)

R

where I is the integrated fluorescence intensity and

I)

∫0∞ I(ν¯ ) dν¯

A is the absorbance, and n is the refractive index of the solvent. The index R refers to the reference fluorophore. Pyrene was chosen as the reference fluorophore of known quantum yield, Q ) 0.32.20 The emission of compound 3 and pyrene was excited at 334 nm and the fluorescence spectra of both fluorophores were recorded in a wavelength range of 338-480 nm. 2.6. Fluorescence Quenching Measurements. Two different series of quenchers were used in the experiments: doxyl-spinlabeled molecules (CSL, 5-SASL, 12-SASL, or 16-SASL) or brominated carboxylic acids (2-Br, 11-Br, or 16-Br). Liposomes containing EYPC, compound 3 (c3 ) 1 µM) and one of the quenchers were prepared according to procedure given in the Materials and Methods (section 2.4). A phosphate buffer of pH 9 were used in the preparation. The final concentration ratio between EYPC and the quencher in the sample was 85:15. The fluorescence emission spectra were measured using an excitation wavelength of 320 nm. The spectra were recorded in a wavelength range of 340-500 nm. At least six samples for each quencher were prepared and the presented results are the averaged values. 2.7. Steady-State Fluorescence Anisotropy Measurements. The anisotropy measurements were recorded in the L-format method on the SLM-AMINCO 8100 Instruments spectrofluorimeter equipped with automatic polarizers. The steady-state anisotropy (r) was calculated according to the equation19

r)

Ivv - GIvh Ivv + 2GIvh

(2)

where I is the fluorescence intensity, and two subscripts refer to the settings of the excitation and emission polarizers, respectively. v and h denote the vertical and horizontal orienta-

tion, respectively. G is an instrumental correction factor, which takes into account the sensitivity of the monochromator to the polarization of light. The G-factor can be easily determined according to the equation19

G)

Ihv Ihh

(3)

G-factors were measured individually for each sample and automatically corrected anisotropy values were obtained. The fluorescence anisotropy of compound 3 (c3 ) 1.7 × 10-5 M, χ3 ) 0.04) in liposomes without and with 5, 10, 15, 20, 25, 30 mol % of Chol was measured at 20 °C. Samples were excited with vertically polarized light at 345 nm and emitted light was analyzed at 360 nm. Due to scattering of the emitted light by the liposome suspension, the interference filter (λmax ) 365 nm) was used in the experiments. The measurements were repeated 10 times for each sample and automatically averaged. Moreover, the effect of turbidity on the decrease of the fluorescence anisotropy of 3 was investigated. The initial concentrations of 3 and lipid in solution were 1.6 × 10-4 M and 2.5 mg/mL, respectively. The initial solution was diluted several times and the anisotropy was measured after each addition of water to the cell. 2.8. Cryotransmission Electron Microscopy (cryo-TEM). Three microliters of the sample solution was applied to an electron microscopy grid covered with perforated supporting film. Most of the sample was removed by blotting (Whatman no. 1 filter paper) for approximately 1 s, and the grid was immediately plunged into liquid ethane held at -183 °C. The sample was then transferred without rewarming into a Tecnai SpheraG20 electron microscope using a Gatan 626 cryospecimen holder. The images were recorded at 120 kV accelerating voltage using a Gatan UltraScan 1000 slow scan CCD camera and low dose mode with the electron dose not exceeding 15 electrons per Å2. Typical value of applied underfocus ranged between 1.5 and 2.7 µm. The applied blotting conditions resulted in specimen with thickness varying between 100 to ca. 300 nm. 2.9. Molecular Dynamics Simulations. In these studies, we performed molecular dynamics (MD) simulation of a lipid bilayer composed of 128 POPC molecules, 3806 water molecules, and 4 molecules of compound 3. The initial configuration of the POPC bilayer used in these studies originated from previous work of Tieleman et al.21 Into this structure, four molecules of compound 3 were inserted manually in various positions and orientations inside the hydrocarbon core of the bilayer. Prior to the MD simulations the starting structure was optimized with 100 steps of steepest descent algorithm to remove unfavorable contacts between atoms. We used the standard force-field parameters for DPPC and DOPC molecules,22 where the partial charges were taken from the underlying model description23 to parametrize the POPC molecule. Water was described using the simple point charge (SPC) model.24 For compound 3 we used Gromos force field parameters compatible with the lipids description.25 The MD simulations were performed with GROMACS 4.0.3 software package.26,27 A time span of 95 ns was simulated. A particlemesh Ewald (PME) method was used for electrostatic interactions with the real cutoff of 1.0 nm.28 The bilayer simulation was performed under constant isotropic pressure of 1 bar and a temperature of 298 K. The temperature and the pressure were kept constant using the V-rescale thermostat with t ) 0.129 and the semiisotropic Parinello-Rahman barostat.30 LINCS con-

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Figure 2. Absorption (dotted line), excitation (solid line), and fluorescence emission (dashed line) spectra of 3 in cyclohexane (A) and EYPC bilayer (B): (A) in fluorescence measurements c3 ) 32 µM, λexc ) 320 nm; (B) in absorption measurement c3 ) 310 µM and the mole fraction of 3 was 0.2, in fluorescence measurement c3 ) 32 µM and the mole fraction of 3 was 0.05, λexc ) 320 nm.

straints algorithm was employed for all bonds31 allowing for a 2 fs time step. Visualizations of the trajectory were made with the VMD package.32 Errors were calculated by using the block analysis method as described in ref 33. 3. Results 3.1. Synthesis and Spectral Characterization of 3. 2,6Bis(decyloxy)naphthalene (3), naphthalene with two n-decyl tails attached at the opposite sides of the aromatic ring, was successfully synthesized as is shown in Figure 1. Naphthalene2,6-diol (1) was reacted with 1-bromodecane (2) in anhydrous environment. The solubility of this compound was checked in a few chosen solvents. It was found that compound 3 is well soluble in THF, CHCl3, cyclohexanol, and cyclohexane and completely insoluble in polar solvents, such as water and DMF. The absorption, excitation, and emission spectra of 3 in cyclohexane and embedded into egg yolk phosphatidylcholine (EYPC) liposomes were recorded at different concentrations. Figure 2A shows the absorption spectrum of compound 3 dissolved in cyclohexane. The attachment of two auxochromic groups such as n-decyloxy to the naphthalene ring produces a bathochromic shift (shift to the longer wavelengths). The vibrational bands of the chromophore are well resolved. Beer’s law was obeyed up to the concentration equal to 0.028 M. The molar absorption coefficient at the maximum of the absorption band was determined to be log ε347 ) 3.56. This value is in good agreement with that reported previously for compound 1 in ethanol.34 When excited at 320 nm, compound 3 gives a broad emission band from 340 to 420 nm with a maximum at 368 nm. The emission spectra are less structured, which reveals some structural change of compound 3 in the excited state after excitation. In the case of lipid dispersion the concentration of the lipid was kept constant (1.0 mg/mL) and the amounts of 3 introduced to the system during preparation were varied. The mole fractions, χ3, were in the range between 0.05 and 0.5 (molecular ratio 3:lipid varied in the range from 1:20 to 1:1). We assumed that all amount of the introduced compound 3 entered to membrane during the preparation of SUV and, therefore, the mole fractions of the compounds in the bilayer are the same as the corresponding mole fractions of compounds combined in the volumetric flask during preparation procedure (but the actual composition of the bilayer could be different). The selected UV-vis absorption, excitation, and emission spectra of compound 3 in the liposome dispersion are shown in Figure 2B. Compared to the organic solvent the maxima of both the absorption and emission bands are slightly red-shifted

to the wavelength of 354 and 370 nm, respectively. These red shifts are due to higher polarity of the liposomal environment (see Supporting Information). The absorption and emission spectra of 3 incorporated into the liposomes did not show any changes in the band shape in the examined range of concentration. This can be explained considering that aggregation phenomena of 3 are suspended after incorporation into lipid bilayer. The quantum yields of 3 fluorescence (Q) in a few selected solvents were determined. Pyrene dissolved in cyclohexane was used as a reference of known quantum yield.20 The values of Q are equal to 0.61, 0.52, and 0.07 in cyclohexane, THF, and CHCl3, respectively. The measured values of Q are very high, showing that compound 3 can fluoresce effectively in apolar environment. As expected, the fluorescence quantum yield in chloroform is very low due to the presence of chlorine atoms in the solvent molecules. 3.2. Cryo-TEM Observation. The penetration of hydrophobic molecules into the vesicle bilayer affects the vesicle stability and its morphology.35 There are several effects which can influence the vesicle morphology during the process of solubilization: (i) increase of the vesicle size, (ii) reorganization into a lamellar structure, or (iii) disintegration of the bilayer after addition of a too high amount of solutes. We incorporated compound 3 into the lipid bilayer in the mole fraction as high as 0.5 (3 to lipid ratio of 1:1), so it is important to check the morphology of the objects formed in the dispersion. To visualize the objects obtained after sonication of such dispersion, we used the cryo-TEM microscopy technique. This technique allows the least perturbing and direct imaging of the hydrated sample. Figure 3 shows the cryo-TEM micrograph and the size distribution profile of the liposomes containing compound 3. As can be seen, the unilamellar vesicles displaying the good spherical shape are present in the dispersion. That is clear evidence that introduction of compound 3 up to χ3 equal to 0.5 into the lipid membrane does not perturb the process of liposome formation. The average diameter of the vesicles observed on the micrographs is 27 ( 14 nm. Similar size distribution for the sonicated EYPC liposomes was determined using dynamic light scattering measurements.36 That kind of liposomes is commonly named as small unilamellar vesicles (SUV). 3.3. Fluorescence Quenching Studies. Information on vertical localization of the molecule inside the lipid membrane is very important. Fluorescence quenching study is one of the simplest ways to obtain qualitative and quantitative information regarding the relative vertical localization of molecule studied



Figure 3. Cryo-TEM micrograph and the diameter profiles of the EYPC liposomes containing mole fraction of 3 equal to χ3 ) 0.5 obtained by sonication. The bar corresponds to 100 nm.

Figure 4. Quenching effect of lipids having quenching moieties attached at different positions on the fluorescence of compound 3 embedded into EYPC liposomes: (A) doxyl-spin-labeled molecules and (B) bromo derivatives of carboxylic acids.

in a bilayer. Comparison of the extent of fluorescence quenching by two lipid-bound quenchers that are located at known, different vertical depths in the bilayer can provide information regarding the vertical depth of the fluorophore in a bilayer. Two different series of the quenchers were used in our studies: doxylspin-labeled molecules, such as 3β-doxyl-5R-cholestane (CSL) and doxyl-labeled stearic acids (5-SASL, 12-SASL, and 16SASL) and bromo derivatives of carboxylic acids, such as 2-bromohexadecanoic acid (2-Br), 16-bromohexadecanoic acid (16-Br), and 11-bromoundecanoic acid (11-Br). The chemical structures of the quenchers and the approximate localizations of quenching moieties (nitroxide and bromide) across the phosphatidylcholine membrane are presented in Figure s3 (Supporting Information). Two compounds, CSL and 2-Br, have quenching moieties that are located in region II of the bilayer. The doxyl group of 5-SASL is located at the interface between region II and I. The quenching groups of the rest of the quenchers are immersed deeper in the membrane (region I). Liposomes containing compound 3 and one of the quenchers at the concentration [lipid]:[quencher] ratio of 85:15 were prepared. The reduction of fluorescence intensity compared to that of the liposomes without quencher was measured. The results are shown in Figure 4. The largest quenching effect was observed for the CSL and 5-SASL from the series of doxylspin-labeled lipids and 2-Br from the series of brominated of carboxylic acids. These findings suggested that the naphthalene ring of compound 3 should be localized in region II of the bilayer. To obtain a more quantitative estimation of the vertical position of the fluorophore in the membrane, the parallax method was applied.37,38 The method allows calculating the vertical depth

of the fluorophore in the bilayer by comparing the extent of quenching that is observed with two lipid-bound quenchers that are located at known, different vertical depths in the bilayer. The distance from the center of the bilayer (Zef) can be calculated by using the equation37

(

)

F1 1 - L221 ln -πC F2 Zef ) + Lc1 2L21

(4)

where F1 and F2 are the fluorescence intensities in the presence of the quencher located at a shallow and deep position, respectively, Lc1 is the distance from the center of the bilayer to the shallow quencher, L21 is the difference in depths of the two quenchers, and C is the concentration of the quencher in molecules per unit area of the bilayer surface. We have used the results obtained for four nitroxide-labeled molecules. The localizations of the quenching moieties of the spin-labeled stearic acids in the bilayer were determined previously by MD simulations.39 The distance of the nitroxide group for 5-SASL, 12-SASL, and 16-SASL from the center of the bilayer was calculated to be 12.7 ( 2.2, 10.2 ( 2.3, and 8.6 ( 2.9 Å, respectively. The position of CSL in the bilayer has not been examined previously. Therefore, we assumed that the depth of the nitroxide group of CSL is similar to the depth of the OH group of the cholesterol molecule. According to the finding by Ro´g and Pasenkiewicz-Gierula, such group is located in the region of the PC phosphate groups.40 The vertical distance from the center of the bilayer of the group was estimated to be 16 Å.

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Figure 5. Fluorescence anisotropy of compound 3 as a function of concentration of cholesterol inside the EYPC bilayers.

Since the largest quenching effect was observed for CSL and 5-SASL, this pair of quenchers was chosen to determine the vertical position of compound 3 in the EYPC membrane, Zef. Our calculations have indicated that the naphthalene ring is located at ca. 14 Å from the center of the bilayer. 3.4. Fluorescence Anisotropy Measurements: Effect of Cholesterol. The measured fluorescence anisotropies of biological samples can be lower than the actual values for trivial reasons, like light scattering and reabsorption.19 Especially, when one is working with the suspension of liposomes, which are often turbid, the scattering of both the incident light and the emission photons can occur. Each scattering event is thought to decrease the fluorescence anisotropy. Therefore, we have investigated the effect of the turbidity of a SUV dispersion on anisotropy values. This was accomplished by dilution of the sample. The anisotropy of compound 3 enclosed into phospholipid bilayer was measured after each addition of water to the cell. It was shown (Supporting Information, Figure s2) that in the diluted dispersions the value of anisotropy is higher than in the more concentrated ones. In further anisotropy experiments we used the concentration of lipid equal to 0.25 mg/mL to eliminate reabsorption and the interference filter (λmax ) 365 nm) to eliminate the scattered light. The value of limiting anisotropy, r0, was determined for 3 enclosed in PMMA thin film at room temperature. The polymer matrix was chosen as a material which completely stops movements of the probe.41 The limiting anisotropy obtained in

Kepczynski et al. this way was 0.312 ( 0.002. This value, if the depolarizing factors are completely absent, gives an angle of 22.5° between the absorption and emission dipoles (r0 ) 0.4 corresponds to an angle of 0°). The observed anisotropy in a vitrified solution is a product of the loss of anisotropy due to angular displacement of the emission dipole relative to the absorption dipoles. We have also investigated the changes of anisotropy values by using various excitation wavelengths. The values of r0 were constant within experimental error across the wavelength of absorption band. Cholesterol (Chol) is one of the main components of the animal cell membranes, which is naturally present at high concentrations. It is well-known that Chol increases the apparent microviscosities (reduces fluidity) of membranes in the liquid phase.42,43 Therefore, we used this additive to modify the properties of the EYPC membrane in the anisotropy measurements. Figure 5 presents data obtained for 3 incorporated into the phospholipid bilayers without and with 5, 10, 15, 20, 25, and 30 mol % of Chol. Figure 5 shows that the fluorescence anisotropy for compound 3 incorporated into SUV increases with increasing Chol content in the membrane. The anisotropy of the probe fluorescence is proportional to the microviscosity of environment. The anisotropy values indicate that the 3 molecules in EYPC membranes experience motional restriction when Chol is introduced. 3.5. Molecular Dynamics Simulations. Our simulations were carried out on a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer as a good model of the EYPC membrane (see the fatty acid makeup in the Materials and Methods section). The hydrated membrane consisted of 64 POPC molecules in each leaflet and a ratio of 30 water molecules per lipid. The most important parameter describing the structure of the bilayer is the area per lipid molecule. That parameter was calculated simply by dividing the 〈x-y〉 plane projection of the mean area of the leaflet by half the number of lipid molecules in the bilayer. The average area per lipid is 0.651 ( 0.004 nm2. Time development of the area per lipid (data not shown) is stable, which results from the long equilibration of the system in the previous simulations (120 ns). An experimental estimate of the area per lipid for fully hydrated POPC is 0.64 ( 0.01 nm2,44 which is in a very good agreement with the simulated value. Position. The four molecules of compound 3 were initially placed at different positions and different orientation inside the membrane, as shown in Figure 6A. Two molecules were situated

Figure 6. Snapshots of the simulated system: initial configuration (A), the configuration at t ) 50 ns (B), and the configuration at t ) 95 ns (C). The four 3 molecules are shown in CPK representation in orange. POPC lipids are shown as sticks and colored as follows: blue, choline; magenta, phosphate; green, glycerol; cyan, hydrocarbon chain. Water molecules are displayed as small spheres with oxygens and hydrogens in red and white, respectively.


Figure 7. Trajectories of the Z coordinate (along bilayer normal) of the ring center of mass of the four compound 3 molecules (green, blue, black, and magenta lines). The center of mass positions of the carbonyl groups of the acyl chains of the POPC lipid are also shown (red line).

with the naphthalene rings close to the center of the membrane and with the decane chains sticking out in the opposite directions one in each leaflet. Two remaining molecules were placed with the rings immersed in the polar region or located close to that region and the tails directed to the lipid layer. The system was then simulated for ca. 95 ns. Figure 7 shows the trajectory of the ring mass center of compound 3 along the membrane normal in the POPC membrane during the simulation run. The molecule with the naphthalene ring in the interfacial region (green line) moved into the lipid bilayer and occupied a position below the carbonyl groups within the first 2 ns. Two molecules (blue and black lines), which were placed in the center of the bilayer, in the period of 10-40 ns move away from the center to the region of the hydrocarbon core close to the carbonyl groups, and stay there for the rest of the simulation. Thus, over the last 25 ns the naphthalene rings of all the molecules of compound 3 occupy positions below the carbonyl groups, as shown in Figure 6C, and do not display any tendency to stay in the center of the lipid bilayer. Figure 7 shows that one of the molecules of 3 migrates between the leaflets of the membrane (green line) and the other one undergoes an unsuccessful migration (the molecule migrates from the position close to the interphase toward the bilayer center and returns to the same interface, magenta line). To elucidate the position of molecule 3 with respect to the lipid molecules, we have calculated the mass density profiles across the membrane. Figure 8 shows the average mass density profiles along the direction normal to the bilayer surface (the Z axis) for the various lipid groups and compound 3 molecules. The density profiles of the lipid groups were averaged over the whole time of the simulations, while that for compound 3 were averaged over the last 20 ns of the trajectories (the profile shown in Figure 8 is multiplied by a factor of 10 to be more visible). The reason for using only the last 20 ns of the trajectory for compound 3 is the migration of the molecules between layers in the earlier stage of the simulation. Thus, for clarity of the presentation we use the part of the trajectory where all molecules show stable behavior. Figure 8 shows that the naphthalene ring of compound 3 occupies the region between the glycerol groups and the double bond in the acyl chains of the lipid molecules. Orientation. The orientation of the molecules 3 inside the POPC membrane should be analyzed in comparison with the orientation of the lipid alkyl chains. The average tilt angle of the palmitoyl chains was calculated from the cosine of the angle


Figure 8. Mass density profile of the naphthalene rings and the various groups along the bilayer normal. For the POPC bilayer, the density profiles were averaged over all the trajectories. For the naphthalene rings the density profiles were averaged over the last 20 ns of the trajectories and multiplied by 10 for better comparison. The profiles are colored as follows: red line, water; blue line, choline; magenta line, phosphate; green line, glycerol; cyan line, hydrocarbon chain, gray line, double bond; black line, CH3 groups; orange line, naphthalene rings.

between the bilayer normal and the average vector linking the carbon atom at the end of the palmitoyl chain and the carbonyl carbon atom of the same chain. The average tilt angle of the palmitoyl chains in the POPC bilayer increased slightly from 31° in the pure membrane to 36° in the presence of compound 3. The orientation of the aromatic ring of the molecules 3 relative to the bilayer normal can be described by two parameters: the angle θlong between the naphthalene ring long axis (defined as the vector between atoms C1 and C6, see also Figure 9A) and the membrane bilayer normal (the Z axis), and the angle θring between the vector normal to the naphthalene ring and the bilayer normal. The normalized distributions of θlong and θring for all 3 molecules in the POPC bilayer for the last 20 ns of the trajectories are shown in Figure 9, B and C. One can see that the distribution of θlong has two distinctive maxima, the more intense one at around 90° and a smaller peak close to 30°. Two Gaussian functions were fitted and the best fit was obtained for the maximum positions of the Gaussian functions at 33° ( 18° and 93° ( 20°. Thus, compound 3 prefers the orientation with their long axis parallel to the plane of the membrane. This favored orientation can be related to the presence of two oxygen atoms, O11 and O22, in the chemical structure of the compound. The peak at the lower angle indicates that a substantial portion (about 35%) of the molecules adopt the alignment of the long axis parallel to the alkyl chains of the bilayer. The distribution of θring is very wide, showing that the ring plane has a great orientational freedom. The vector normal to the plane of the aromatic ring makes an average angle of 77 ( 35° with the bilayer normal. Therefore, a vector parallel to the plane of the aromatic ring should make an average angle of 13° with the bilayer normal, because these vectors are orthogonal. Thus, the plane of the aromatic ring of 3 aligns almost perpendicularly to the plane of the membrane, as can be seen in Figure 6, B and C. The other information is the orientation of the decyl hydrocarbon chains attached to the naphthalene ring. The tilt angle of the alkyl chains of compound 3 was measured as an angle (θchain) between the vector linking the first and last carbon of the same chain (the chain vector) and the bilayer normal (Z axis) (see Figure 9A). Figure 9D shows the distribution of the

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Figure 9. (A) Definition of the long axis, the vector normal to the naphthalene ring, and the vector of decyl chains of the compound 3. (B-D) Probability distributions P(θ) of the angles between the long axis (θlong, B), the vector normal to the aromatic ring (θring, C), and the chain vector (θchain, D) relative to the bilayer normal for all 3 molecules over the last 20 ns of the trajectories. Dashed lines show the fitted Gaussian curves and solid lines are the sum of fitted curves.

angle θchain for all the 3 molecules in the POPC bilayer for the last 20 ns of the trajectories. The distribution shows two separate maxima at the angles of 50 ( 26° and 125 ( 30°. These two angles represent basically the same direction of the alkyl chain as measured for the molecules in the upper and the lower leaflet of the bilayer, respectively. Since, in the presence of compound 3, the average angle of the palmitoyl chains in the POPC bilayer to the bilayer normal is 36°, the hydrocarbon chains of 3 are tilted at ca. 14-20° to the lipid acyl chains. Generally, all presented distributions are rather broad showing that compound 3 has great orientational freedom in the POPC bilayer. 4. Discussion 2,6-Bis(decyloxy)naphthalene (3) was synthesized and its interaction with a lipid membrane was studied using experimental and computer simulations methods. The main objective to study such a compound was to explore the effect of attachment of long hydrocarbon tails on the behavior of small organic molecule inside the lipid membrane. Compound 3 has in its structure the naphthalene ring, which plays a role of a fluorophore, and two long hydrocarbon chains attached to the opposite sides of the aromatic ring. The hydrocarbon tails can be incorporated in the lipid bilayer. The structure of the studied compound makes it possible to adopt various conformations inside the bilayer. Therefore, the position and conformation of compound 3 inside the phosphatidylcholine (PC) bilayer is very important information for further consideration.

One could expect at least two different conformations of compound 3 embedded in PC membrane and, consequently, two different locations of the aromatic ring inside the lipid bilayer. One conformation (hereafter defined as conformation A) of the molecule of 3 is that with the naphthalene ring located near the polar region (region II) and the tails being parallel to the lipid chains. That conformation is similar to the lipid alignment in membrane, so it can be called “phospholipid-like” conformation. The second conformation (conformation B) is with the 3 ring located in the center of membrane (region I) and with both decane chains sticking out in opposite directions one in each leaflet. MD simulations are the most accurate method to determine which conformation of compound 3 is more stable. Four different conformers of compound 3 were placed inside the POPC bilayer, as shown in Figure 6A. Two molecules were situated with the naphthalene rings close to the center of the membrane and the decane chains sticking out in opposite directions, one in each leaflet (conformation B). Two remaining molecules were placed with the rings immersed in the polar region or close to that region and the tails directed to the lipid chains (conformation A). During the simulation, the molecules in conformation B moved to the carbonyl region and adopted conformation A. At the end of a 50 ns run, three molecules of compound 3 reoriented to adopt a position with the long axis of the aromatic ring parallel to the lipid bilayer surface and one molecule adopted the conformation B, with the ring in the center of the membrane, as shown in Figure 6B. At the end of a 95 ns run, all the molecules of compound 3 adopted the conformation

2,6-Bis(decyloxy)naphthalene Inside Lipid Bilayer A. Therefore, the preferred location of the naphthalene ring inside the membrane is that in the region between the carbonyl groups and the double-bond groups of the alkyl chains of the lipids, which is a region of high tail density and low free volume compared to the center of the bilayer. The rings are oriented in such a way that the long axes of the aromatic ring are oriented mostly parallel to the lipid bilayer or parallel to the lipid chains, whereas the ring plane is almost perpendicular to the membrane surface. From Figure 9D, one can see that the tails show a preferred alignment with the chain vector oriented more or less parallel to the bilayer normal with a tilt of (20°. So the tails tend to arrange themselves parallel to the acyl chains, thereby minimizing the perturbation of the lipid chains. The arrangement of the molecule 3 with the naphthalene rings being in region II of the lipid bilayer and two hydrocarbon chains directed into the most hydrophobic part of the membrane can be explained considering several reasons. The most important reason is the presence of two electronegative oxygen atoms as the substituents of the aromatic ring. In our simulations these atoms bear partial charges of -0.222. The charges on the ether oxygens would force them to align with the ester oxygen of the lipids. The effect of altering the partial charges in indole and benzene was previously simulated by Norman and Nymerer.16 It has been shown that uncharged molecules of both compounds reside in the hydrocarbon core. However, the appearance of net charges on the molecules resulted in their significant exclusion from the center of the bilayer and stabilization within the interfacial region. The other reason is related to the entropy changes. In the middle region of the membrane the acyl chains are highly disordered, resembling liquid hexane.45 Presence of rigid molecules, such as naphthalene would force the acyl chains to arrange themselves around that molecule, and would reduce the mobility of the chain ends. That would result in entropy decrease. Thus, it is more favorable for the system to accommodate naphthalene rings in the region closer to the headgroup, where the chain order is higher. Such entropic reasons were previously proposed as an explanation for partitioning of pyrene in region II.17 Both above-mentioned reasons can be considered as driving forces for escape of the naphthalene fluorophore from the center of the bilayer. On the contrary, the hydrocarbon tails have a tendency to partition preferentially in the center of the bilayer. As was shown previously, this process is driven almost entirely by a favorable entropy change, consistent with the hydrophobic effect.15 The computer simulations were complemented with experimental studies on the interaction of compound 3 with EYPC liposomes. First, the shape and position of absorption and fluorescence excitation spectra can serve as evidence that compound 3 has partitioned into the lipid membrane. The spectra of compound 3 in the presence of SUV’s are very similar to those in organic solvent. Moreover, the molecules of the investigated probe in the lipid bilayer were isolated and no evidence of aggregation was observed. As was shown previously, the penetration of the hydrophobic molecules into the vesicle bilayer affects the vesicle stability and its morphology.35 The influence of the solute on the vesicular structure depends on its amount and nature. Solubilization of aromatic molecules, e.g., toluene, into the catanionic vesicle bilayer leads to enlargement and deformation of the vesicles and, finally, after introducing it at too high concentration, to formation of the lamellar structure. However, the catanionic vesicles still remain in a unilamellar structure upon the solubilization similar amount of octane. The various effects of solubilization of hydrocarbons on the catanionic vesicles were accredited to the positions which


Figure 10. Snapshots taken after different times of simulation showing migration of compound 3 from one leaflet to another. Compound 3 is shown in CPK representation in orange. Acyl chains of POPC lipids are shown as cyan sticks, and phosphate groups are shown as magenta spheres. The water molecules are displayed in red and gray.

they occupy in the bilayer. As compound 3 has in its structure both the aromatic and hydrocarbon groups, it was reasonable to expect some effects of the naphthalene group on the morphology of the liposomes after embedding as much as 0.5 mole fraction of that compound into the membrane. Direct visualization of the system by means of cryo-TEM has demonstrated that such a high concentration of 3 has no effect on the formation of vesicular structures. The liposomes formed by sonication possess almost ideal spherical shape. The position of the naphthalene rings of compound 3 near the headgroups seems to be the favorable location inside the lipid membrane. We used the fluorescence quenching methods to confirm that location. The quenching experiments involved lipids having in the chemical structure quenching moiety attached at the different vertical positions. These measurements showed that the most effective quenching process was achieved when the quenching groups were located in region II or at the interface between regions II and I. Very interesting results which were obtained from the MD simulations indicated the possibility of a translocation (flip-flop) of compound 3 from one monolayer to the other. The molecular mechanism of such transition is related to assuming of conformation B by molecule 3, as shown in Figure 6B. The overall process of transmembrane translocation of compound 3 is visualized in Figure 10 (an animation of the transmembrane translocation of molecule 3 made from the trajectory in wmv

15492 J. Phys. Chem. B, Vol. 114, No. 47, 2010 format is available in the Supporting Information). Initially, the molecule has the lipidlike conformation (conformation A) and is located in one of the monolayers (Figure 10, 2 ns). After about 40 ns, one of the tails of the molecule starts moving toward the second monolayer (Figure 10, 45 ns). That movement is mainly driven by thermal fluctuations. Diffusion of the tail to another leaflet draws the naphthalene ring into the center of the bilayer (Figure 10, 49 ns). Finally, the whole molecule gains the conformation B (Figure 10, 54 ns). The molecule stays in this transient conformation for about 5-6 ns (Figure 10, 60 ns). The aromatic ring tends to escape from the center of membrane to the more preferential position near the carbonyl region, pulling another tail. That eventually leads to transition of the molecule 3 to the second monolayer, together with assuming the conformation A (Figure 10, 65 ns). But the naphthalene ring can also return to the starting monolayer, which was observed as the unsuccessful migration. Gurtovenko and Vattulainen46 have previously studied a transmembrane translocation of lipid molecules in the bilayer. Flip-flop of lipids within a membrane not containing any specific membrane proteins is normally very slow (from several hours to several days), because it requires the polar headgroup of a lipid to traverse the hydrophobic core of the membrane. Using the MD simulations, these authors have shown that the appearance of a water pore, spanning a phospholipid membrane, leads to a diffusion of the polar headgroups along the pore. Therefore, this translocation occurs with rotation of the whole lipid molecule. The mechanism of translocation of compound 3 through the lipid membrane is quite different from that demonstrated for flip-flop of lipids. We have showed that, due to diffusion of the one decane tail to the opposite leaflet, the molecule 3 first unfolds itself and assumes the “open” conformation, with the ring placed between the leaflets and the tails sprawled to the opposite membrane leaflets, then it folds up to the lipidlike conformation in the opposite monolayer. In the case of our compound the migration throughout the lipid bilayer is facilitated, because all parts of compound 3 are hydrophobic. However, there is an important question regarding the effect of attachment of long hydrocarbons tails on the possibility of the translocation of compound 3. There are no data in the literature concerning migration of free naphthalene between the leaflets of membrane. However, no tendency for the free molecules of pyrene to move from one monolayer leaflet of the membrane across the hydrophobic core to the other half was previously reported.17 Therefore, we believe that the presence of two hydrocarbon tails is rather important for translocation of compound 3 across the bilayer. Thus, the attachment of the long tails to the organic molecule can facilitate the passive transport of that molecule through the artificial membrane and most likely also cell membrane. Various fluorescence probes are often used as powerful tools to monitor the dynamics and organization of modified membranes.12,47-49 Embedding of a probe into the lipid bilayer and observation of its fluorescence properties, such as fluorescence depolarization or excimer formation, is a very convenient method to obtain information regarding the membrane properties. Compound 3 can serve as a rotational molecular probe for investigation of lipid membrane properties in the acyl side chain region. The probe was constructed in the manner which helps to limit the perturbation of the membrane organization. Two long hydrocarbon chains were attached to the naphthalene molecule, a relatively small fluorophore. The chains of 3 can penetrate down to the deeper nonpolar part of the liposome. They are saturated hydrocarbon chains, so they are expected

Kepczynski et al. not to perturb the local environment. The naphthalene ring enables monitoring the changes in the rotation of the molecule by using the anisotropy measurements. To check the usefulness of compound 3 as a reporter of membrane fluidity, we have used Chol to modify of the EYPC membrane properties. Using MD simulation study, it was shown that Chol added to the membrane induces the ordering of the lipid acyl chains (so-called ordering effect) by inhibition of the trans-gauche transitions.50 Induced in that way, tightening of the lipid packing (condensation effect) in the bilayers affects their fluidity, viscosity, and lateral diffusion. Thus, the presence of Chol in the membranes being in liquid phase increases the apparent microviscosities (reduces fluidity). We have previously shown that the microviscosity of the EYPC membrane increases 1.4 times when concentration of Chol increases to 30 mol %.12 Therefore, the introduction of Chol into the EYPC membrane should have a strong influence on the rotational freedom of compound 3 in the lipid bilayer. The effect of embedding of various amounts of Chol (0-30 mol %) into the EYPC membranes was investigated by the fluorescence anisotropy measurements of 3. Indeed, the anisotropy of 3 increases with increasing of Chol concentration in the membrane (Figure 5). Thus, the phospholipid bilayer which contains 5 mol % and more of Chol behaves as a barrier for rotations of 3. However, the increase in the anisotropy of 3 with the Chol concentration is not linear. The r value rises strongly up to 10 mol % of Chol, and then the slope decreases abruptly. Finally, for the Chol concentration of 30 mol % a decrease of the r value was observed. These observations can be explained taking into account a phase diagram of the phospholipid-Chol system. Binary phase diagrams for mixtures of various phospholipids with cholesterol were previously studied experimentally.51 For the POPC-Chol system at 20 °C three concentration regions can be distinguished. Below 8 mol % of Chol the system is in the liquid disordered phase (LR), in the range of 8-25 mol % LR and the liquid ordered phase (Lo) coexist, and at the concentration greater than 25 mol % the system is in the Lo phase. Thus, measurements of the fluorescence anisotropy of compound 3 could find application in the studies of lipid membrane properties in the acyl side chain region and in determination of the phase boundaries in the lipid-Chol binary systems. 5. Conclusion We have synthesized the derivative of naphthalene with two hydrocarbon chains attached at the opposite sites of the aromatic ring (2,6-bis(decyloxy)naphthalene, 3). This compound was incorporated into phosphatidylcholine (PC) vesicles and its behavior was studied. To determine the preferred localization and orientation of 3 molecule in the lipid bilayer, we have performed MD simulations. The results from the MD simulations provide us with the information on the dynamics and structural features of the system. The naphthalene fluorophore in compound 3 was found to be solubilized preferentially in the highly ordered upper acyl chain region near the lipid headgroups, and the hydrocarbon chains of 3 are directed to the center of the bilayer. The computer simulations indicated that compound 3 can migrate from one leaflet of the membrane to the other. The molecular mechanism of such transition involves the change of conformation of compound 3 with naphthalene ring located between the leaflets and tails sprawled to opposite membrane leaflets. Using fluorescence quenching techniques, the localization of 3 in the phospholipid bilayer was

2,6-Bis(decyloxy)naphthalene Inside Lipid Bilayer confirmed. We have demonstrated also that the probe 3 can be used for estimation of the fluidity of liposomes modified with cholesterol. Acknowledgment. This project was operated within the Foundation for Polish Science Team Programme cofinanced by the EU European Regional Development Fund, PolyMed, TEAM/2008-2/6. T.R. thanks the Academy of Finland for financial support. J.B. acknowledges the support of the Czech Grants LC535, MSM0021620806, and AV0Z50110509. Supporting Information Available: Snapshot and mass density profiles of the POPC bilayer. Effect of the dilution on the anisotropy of 3 in EYPC liposomes with cholesterol. The chemical structures of the quenchers used in the fluorescence quenching studies and their approximate localizations in a bilayer. Time-resolved fluorescence measurements. Stern-Volmer plots for KI and CuSO4 quenching of compound 1 and 3. An animation of the transmembrane translocation of molecule 3 made from the trajectory in wmv format. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Sadzuka, Y.; Nakade, A.; Tsuruda, T.; Sonobe, T. Study on the characterization of mixed polyethyleneglycol modified liposomes containing doxorubicin. J. Controlled Release 2003, 91, 271–280. (2) Soni, V.; Kohli, D. V.; Jain, S. K. Transferrin coupled liposomes as drug delivery carriers for brain targeting of 5-florouracil. J. Drug Target 2005, 13, 245–250. (3) Howell, B. A.; Chauhan, A. Interaction of Cationic Drugs with Liposomes. Langmuir 2009, 25, 12056–12065. (4) Howell, B. A.; Chauhan, A. Current and Emerging Detoxification Therapies for Critical Care. Materials 2010, 3, 2483–2505. (5) MacCallum, J. L.; Tieleman, D. P. Interactions between small molecules and lipid bilayers. Curr. Top. Membr. Transport 2008, 60, 227– 256. (6) Kepczynski, M.; Karewicz, A.; Gornicki, A.; Nowakowska, M. Interactions of porphyrin covalently attached to poly(methacrylic acid) with liposomal membranes. J. Phys. Chem. B 2005, 109, 1289–1294. (7) Nawalany, K.; Kozik, B.; Kepczynski, M.; Zapotoczny, S.; Kumorek, M.; Nowakowska, M.; Jachimska, B. Properties of Polyethylene Glycol Supported Tetraarylporphyrin in Aqueous Solution and Its Interaction with Liposomal Membranes. J. Phys. Chem. B 2008, 112, 12231–12239. (8) Kaiser, R. D.; London, E. Location of Diphenylhexatriene (DPH) Derivatives Within Membranes: Comparison of Different Fluorescence Quenching Analyses of Membrane Depth. Biochemistry 1998, 37, 8180– 8190. (9) Mukherjee, S.; Raghuraman, H.; Chattopadhyay, A. Membrane localization and dynamics of Nile Red: effect of cholesterol. Biochim. Biophys. Acta 2007, 1768, 59–66. (10) Bronshtein, I.; Afri, M.; Weitman, H.; Frimer, A. A.; Smith, K. M.; Ehrenberg, B. Porphyrin depth in lipid bilayers as determined by iodide and parallax fluorescence quenching methods and its effect on photosensitizing efficiency. Biophys. J. 2004, 87, 1155–1164. (11) Saxena, R.; Shrivastava, S.; Chattopadhyay, A. Exploring the organization and dynamics of hippocampal membranes utilizing pyrene fluorescence. J. Phys. Chem. B 2008, 112, 12134–12138. (12) Kepczynski, M.; Nawalany, K.; Kumorek, M.; Kobierska, A.; Jachimska, B.; Nowakowska, M. Which physical and structural factors of liposome carriers control their drug-loading efficiency. Chem. Phys. Lipids 2008, 155, 7–15. (13) Sonnen, A. F.-P.; Bakirci, H.; Netscher, T.; Nau, W. M. Effect of Temperature, Cholesterol Content, and Antioxidant Structure on the Mobility of Vitamin E Constituents in Biomembrane Models Studied by Laterally Diffusion-Controlled Fluorescence Quenching. J. Am. Chem. Soc. 2005, 127, 15575–15584. (14) Xiang, T.-X.; Anderson, B. D. Liposomal drug transport: a molecular perspective from molecular dynamics simulations in lipid bilayers. AdV. Drug DeliVery ReV. 2006, 58, 1357–1378. (15) MacCallum, J. L.; Tieleman, D. P. Computer simulation of the distribution of hexane in a lipid bilayer: spatially resolved free energy, entropy, and enthalpy profiles. J. Am. Chem. Soc. 2006, 128, 125–130. (16) Norman, K. E.; Nymeyer, H. Indole localization in lipid membranes revealed by molecular simulation. Biophys. J. 2006, 91, 2046–2054.

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JP103753F

Behavior of 2, 6-Bis (decyloxy) naphthalene Inside Lipid Bilayer

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